Mechanical Activation of Valvular Interstitial Cell Phenotype: A Dissertation

During heart valve remodeling, and in many disease states, valvular interstitial cells (VICs) shift to an activated myofibroblast phenotype which is characterized by enhanced synthetic and contractile activity. Pronounced alpha smooth muscle actin (αSMA)-containing stress fibers, the hallmark of activated myofibroblasts, are also observed when VICs are placed under tension due to altered mechanical loading in vivo or during in vitro culture on stiff substrates or under high mechanical loads and in the presence of transforming growth factor-beta1 (TGF-β1). The work presented herein describes three distinct model systems for application of controlled mechanical environment to VICs cultured in vitro. The first system uses polyacrylamide (PA) gels of defined stiffness to evaluate the response of VICs over a large range of stiffness levels and TGF-β1 concentration. The second system controls the boundary stiffness of cell-populated gels using springs of defined stiffness. The third system cyclically stretches soft or stiff two-dimensional (2D) gels while cells are cultured on the gel surface as it is deformed. Through the use of these model systems, we have found that the level of 2D stiffness required to maintain the quiescent VIC phenotype is potentially too low for a material to both act as matrix to support cell growth in the non-activated state and also to withstand the mechanical loading that occurs during the cardiac cycle. Further, we found that increasing the boundary stiffness on a three-dimensional (3D) cell populated collagen gel resulted in increased cellular contractile forces, αSMA expression, and collagen gel (material) stiffness. Finally, VIC morphology is significantly altered in response to stiffness and stretch. On soft 2D substrates, VICs cultured statically exhibit a small rounded morphology, significantly smaller than on stiff substrates. Following equibiaxial cyclic stretch, VICs spread to the extent of cells cultured on stiff substrates, but did not reorient in response to uniaxial stretch to the extent of cells stretched on stiff substrates. These studies provide critical information for characterizing how VICs respond to mechanical stimuli. Characterization of these responses is important for the development of tissue engineered heart valves and contributes to the understanding of the role of mechanical cues on valve pathology and disease onset and progression. While this work is focused on valvular interstitial cells, the culture conditions and methods for applying mechanical stimulation could be applied to numerous other adherent cell types providing information on the response to mechanical stimuli relevant for optimizing cell culture, engineered tissues or fundamental research of disease states

hMSC response to stretch is unclear due to spreading on static soft gels.

<p>Micrographs of hMSCs cultured statically (left column) and following ∼10% cyclic equibiaxial strain (right column) for 6 hours on soft gels (0.3 kPa, top row) and stiff gels (50 kPa, bottom row). Staining for f-actin (green) and nuclei (blue) shows that hMSCs on soft gels (static and stretched) have unorganized actin fibers whereas cells on stiff gels have more organized actin fibers. Unlike VICs, hMSCs spread well on soft gels and stretch appears to increase the spread area of the cells slightly on stiff gels. Scale bar = 100 µm.</p

Schematic of polyacrylamide gel on flexible silicone membrane under static (A) and stretched (B) conditions.

<p>Top view of a 22 mm diameter collagen-coated gel (∼70 µm thickness) is cast into a 35 mm diameter flexible-bottomed Flexcell™ well (C) and STREX well (C, insert). Image of Flexcell™ well (D) stretched above an Arctangle™ loading post and labeled with retroreflective beads for strain field analysis. Rectangle shows region analyzed in HDM software, arrows point to edge of gel. Scale bars = 10 mm in all panels.</p

Strain field in region of interest is roughly uniform for equibiaxial stretch.

<p>Strain maps for a soft gel (0.3 kPa) undergoing equibiaxial strain in the X (A), Y (B), and XY (shear, C) directions demonstrating relatively homogenous strain and minimal shear within the area of analysis of cell morphology (dashed box). (D) CAD representation of the circular loading platen over which the silicone membrane is stretched by vacuum pressure. Scale bars = 5 mm.</p

Average strain (± SD) within central region used for analysis of cell morphology for equibiaxial stretch (round loading post) and uniaxial stretch (Arctangle™ loading post).

<p>Average strain (± SD) within central region used for analysis of cell morphology for equibiaxial stretch (round loading post) and uniaxial stretch (Arctangle™ loading post).</p

VICs on soft (0.3 kPa) and stiff (50 kPa) gels cultured under static and pure uniaxial stretch conditions (1 Hz, 10% stretch, 6 hrs).

<p>Cells cultured on soft substrates appear to have less realignment with stretch compared to the classic realignment perpendicular to the direction of stretch on the stiff substrates. Scale bar = 100 µm.</p

Strain field in region of interest is roughly uniform for pure uniaxial stretch.

<p>Strain maps for a soft gel (0.3 kPa) undergoing pure uniaxial strain in the X (A), Y (B), and XY (shear, C) directions demonstrating relatively homogenous strain and minimal shear within the area of analysis of cell morphology (dashed box). (D) CAD representation of the Arctangle™ loading platen over which the silicone membrane is stretched by vacuum pressure. Scale bars = 5 mm.</p

When cyclically stretched, cells on stiff substrates reduce spread area whereas cells on soft substrates increase spread area: Area (A) and perimeter (B) of VICs cultured on low (0.3 kPa) and high (50 kPa) stiffness gels subjected to 10% cyclic stretch at 1 Hz for 6 hours (grey bars) or static (black bars) culture.

<p>Shape factor (C) quantifies how rounded a cell is (a shape factor of 1 is perfectly circular, whereas a shape factor of 0 is extremely spread with many extensions). Cells of low and high shape factor are shown in C. Brackets above bars show significance between individual groups (two-way ANOVA, p<0.05).</p